Methods using cre-lox for production of recombinant adeno-associated viruses

ABSTRACT

Methods for efficient production of recombinant AAV are described. In one aspect, three vectors are introduced into a host cell. A first vector directs expression of cre recombinase, a second vector contains a promoter, a spacer sequence flanked by loxP sites and rep/cap, and a third vector contains a minigene containing a transgene and regulatory sequences flanked by AAV ITRs. In another aspect, the host cell stably or unducibly expresses cre recombinase and two vectors carrying the other elements of the system are introduced into the host cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/242,743, filed Feb. 22, 1999, which will issue as U.S. Pat. No.6,274,354, on Aug. 14, 2001, which is a 371 of PCT/US97/15691, filedSep. 4, 1997, which claims the benefit of U.S. Provisional ApplicationNo. 60/025,323, filed Sep. 6, 1996, abandoned.

BACKGROUND OF THE INVENTION

This invention relates generally to production methods for recombinantviruses, and more specifically, to methods of producing recombinantadeno-associated viruses.

Adeno-associated virus (AAV) is a replication-deficient parvovirus, thegenome of which is about 4.6 kb in length, including 145 nucleotideinverted terminal repeats (ITRs). Two open reading frames encode aseries of rep and cap polypeptides. Rep polypeptides (rep78, rep68,rep62 and rep40) are involved in replication, rescue and integration ofthe AAV genome. The cap proteins (VP1, VP2 and VP3) form the virioncapsid. Flanking the rep and cap open reading frames at the 5′ and 3′ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp ofwhich are capable of forming Y- or T-shaped duplex structures. Ofimportance for the development of AAV vectors, the entire rep and capdomains can be excised and replaced with a therapeutic or reportertransgene [B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser,CRC Press, pp.155-168 (1990)]. It has been shown that the ITRs representthe minimal sequence required for replication, rescue, packaging, andintegration of the AAV genome.

When this nonpathogenic human virus infects a human cell, the viralgenome integrates into chromosome 19 resulting in latent infection ofthe cell. Production of infectious virus and replication of the virusdoes not occur unless the cell is coinfected with a lytic helper virus,such as adenovirus or herpesvirus. Upon infection with a helper virus,the AAV provirus is rescued and amplified, and both AAV and helper virusare produced. The infecting parental ssDNA is expanded to duplexreplicating form (RF) DNAs in a rep dependent manner. The rescued AAVgenomes are packaged into preformed protein capsids (icosahedralsymmetry approximately 20 nm in diameter) and released as infectiousvirions that have packaged either + or −ss DNA genomes following celllysis.

AAV possesses unique features that make it attractive as a vector fordelivering foreign DNA to cells. Various groups have studied thepotential use of AAV in the treatment of disease states. Progresstowards establishing AAV as a transducing vector for gene therapy hasbeen slow for a variety of reasons. While the ability of AAV tointegrate in quiescent cells is important in terms of long termexpression of a potential transducing gene, the tendency of theintegrated provirus to preferentially target only specific sites inchromosome 19 reduces its usefulness.

However, an obstacle to the use of AAV for delivery of DNA is lack ofhighly efficient schemes for encapsidation of recombinant genomes andproduction of infectious virions. See, R. Kotin, Hum. Gene Ther.,5:793-801 (1994)]. One such method involves transfecting the rAAV genomeinto host cells followed by co-infection with wild-type AAV andadenovirus. However, this method leads to unacceptably high levels ofwild-type AAV. Incubation of cells with rAAV in the absence ofcontaminating wild-type AAV or helper adenovirus is associated withlittle recombinant gene expression. In the absence of rep, integrationis inefficient and not directed to chromosome 19.

A widely recognized means for manufacturing transducing AAV virionsentails co-transfection with two different, yet complementing plasmids.One of these contains the therapeutic or reporter transgene sandwichedbetween the two cis acting AAV ITRs. The AAV components that are neededfor rescue and subsequent packaging of progeny recombinant genomes areprovided in trans by a second plasmid encoding the viral open readingframes for rep and cap proteins. Overexpression of Rep proteins havesome inhibitory effects on adenovirus and cell growth [J. Li et al, J.Virol., 71:5236-5243 (1997)]. This toxicity has been the major source ofdifficulty in providing these genes in trans for the construction of ausefull rAAV gene therapy vector.

There remains a need in the art for the methods permitting the efficientproduction of AAV and recombinant AAV viruses for use as vectors forsomatic gene therapy.

SUMMARY OF THE INVENTION

The present invention provides methods which permit efficient productionof rAAV, which overcome the difficulties faced by the prior art. Thismethod is particularly desirable for production of recombinant AAVvectors useful in gene therapy. The method involves providing a hostcell with

(a) a cre transgene, which permits splicing out of the rep and cap geneinhibitory sequences that when removed lead to activation of rep andcap;

(b) the AAV rep and cap genes, 5′ to these genes is a spacer which isflanked by lox sites;

(c) a minigene comprising a therapeutic transgene flanked by AAV inverseterminal repeats (ITRs); and

(d) adenovirus or herpesvirus helper functions.

Thus, in one aspect, the invention provides a method for producing arAAV which comprises introducing into a host cell a first vectorcontaining the cre transgene under regulatory control of sequences whichexpress the gene product thereof in vitro, a second vector containing aspacer flanked by lox sites, which is 5′ to the rep and cap genes, and athird vector containing a therapeutic transgene flanked by AAV ITRs.These vectors may be plasmids or recombinant viruses. One of the vectorscan be a recombinant adenovirus or herpesvirus, which can provide to thehost cell the essential viral helper functions to produce a rAAVparticle. However, if all the vectors are plasmids, the cell must alsobe infected with the desired helper virus. The cell is then culturedunder conditions permitting production of the cre recombinase. Therecombinase causes deletion of the spacer flanked by lox sites upstreamof the rep/cap genes. Removal of the spacer allows the rep and cap genesto be expressed, which in turn allows packaging of the therapeutictransgene flanked by AAV ITRs. The RAAV is harvested thereafter.

In another aspect, the invention provides a method wherein a host cellexpressing cre recombinase is co-transfected with a vector carrying aspacer flanked by lox sites 5′ to the rep and cap genes, and a vectorcontaining the therapeutic minigene above. With the provision of helperfunctions by a means described herein, the cell is then cultured underappropriate conditions. When cultured, the cre recombinase causesdeletion of the spacer thus activating expression of rep/cap, resultingin the rAAV as described above.

In yet another aspect, the present invention provides rAAV vectorsproduced by the methods of the invention.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a 1600 bp DNA fragment containinggreen fluorescent protein (GFP) cDNA, an intron and a polyadenylation(pA or polyA) signal useful as a spacer in a vector of the invention.

FIG. 2 is a schematic illustration of a 1000 bp DNA fragment containingthe gene encoding neomycin resistance (neo^(R)) and a polyA usefril as aspacer.

FIG. 3 illustrates a plasmid pG.CMV.nls.CRE, usefil for transfection ofhuman embryonic kidney 293 cells in the method of the invention.

FIG. 4 illustrates a plasmid pAd.P5.Sp.Rep/Cap, useful in the method ofthe invention.

FIG. 5 illustrates the construction of the recombinant adenovirus,Ad.CMV.NLS-CRE, useful in the method of the invention.

FIG. 6A illustrates the structure of the Ad.CAG.Sp.LacZ virus.

FIG. 6B provides the Southern blot analysis of genomic DNA isolated from293 cells infected with the LacZ virus at a m.o.i. of 1 and cut withNotI. The 1000 bp ³²p-NEO spacer was used as a probe. After thedigestion with NotI a 6200 bp restriction fragment (without cre-mediatedrecombination) and/or a 5200 bp restriction fragment (with cre-mediatedrecombination) can be detected.

FIG. 6C provides the Southern blot analysis of genomic DNA isolated from293 cells infected with the LacZ virus at a m.o.i. of 10 and cut withNotI. The 1000 bp ³² P-NEO spacer was used as a probe. After thedigestion with NotI a 6200 bp restriction fragment (without cre-mediatedrecombination) and/or a 5200 bp restriction fragment (with cre-mediatedrecombination) can be detected.

FIG. 6D provides the Southern blot analysis genomic DNA isolated from293 cells infected with the LacZ virus at a m.o.i. of 100 and cut withNotI. The 1000 bp ³²P-NEO spacer was used as a probe. After thedigestion with NotI a 6200 bp restriction fragment (without cre-mediatedrecombination) and/or a 5200 bp restriction fragment (with cre-mediatedrecombination) can be detected.

FIG. 7 illustrates the structure of the Ad.Tre.CMV.GFP.Rep/Cap virus.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for rAAV production using the cre-loxsystem, which overcomes the difficulties previously experienced inproviding efficient production systems for recombinant AAV. The methodof this invention produces rAAV carrying therapeutic transgenes, whichare particularly usefuil in gene therapy applications.

In summary, the method involves culturing a selected host cell whichcontains

(a) a cre transgene

(b) the AAV rep and cap genes, 5′ to these genes is a spacer flanked bylox sites;

(c) a minigene comprising a therapeutic transgene flanked by AAV ITRs;and

(d) adenovirus or herpesvirus helper functions.

The use of the term “vector” throughout this specification refers toeither plasmid or viral vectors, which permit the desired components tobe transferred to the host cell via transfection or infection. By theterm “host cell” is meant any mammalian cell which is capable offunctioning as an adenovirus packaging cell, i.e., expresses anyadenovirus proteins essential to the production of AAV, such as HEK 293cells and other packaging cells. By the term “minigene” is meant thesequences providing a therapeutic transgene in operative associationwith regulatory sequences directing expression thereof in the host celland flanked by AAV ITRs. The term “transgene” means a heterologous geneinserted into a vector.

Desirably, components (a), (b) and (c) may be carried on separateplasmid sequences, or carried as a transgene in a recombinant virus.Alternatively, the cre protein may be expressed by the selected hostcell, therefor not requiring transfection by a vector. For each of thesecomponents, recombinant adenoviruses are currently preferred. However,using the information provided herein and known techniques, one of skillin the art could readily construct a different recombinant virus (i.e.,non-adenovirus) or a plasmid vector which is capable of drivingexpression of the selected component in the host cell. For example,although less preferred because of their inability to infectnon-dividing cells, vectors carrying the required elements of thissystem, e.g., the cre recombinase, may be readily constructed usinge.g., retroviruses or baculoviruses. Therefore, this invention is notlimited by the virus or plasmid selected for purposes of introducing thecre recombinase, rep/cap, or minigene into the host cell.

Desirably, however, at least one of the vectors is a recombinant viruswhich also supplies the helper functions (d) to the cell. Alternatively,the helper functions may be supplied by co-infecting the cell with ahelper virus, i.e., adenovirus or herpesvirus, in a conventional manner.The resulting rAAV containing the minigene may be isolated therefrom.

A. The Cre Transgene

The cre protein is a recombinase isolated from bacteriophage P1 whichrecognizes a specific sequence of 34 bp (loxP). Recombination betweentwo loxP sites (catalyzed by the cre protein) causes, in certain cases,the loss of sequences flanked by these sites [for a review see N. Kilbyet al, Trends Genet., 9:413-421 (1993)]. The sequences of cre areprovided in N. Sternberg et al, J. Mol. Biol., 187:197-212 (1986) andmay alternatively be obtained from other commercial and academicsources. The expression of the cre protein in the cell is essential tothe method of this invention.

Without wishing to be bound by theory, the inventors believe that theexpression of cre recombinase in the host cell permits the deletion ofthe “spacer” DNA sequence residing between the promoter and rep/capgenes in the second vector. This deletion of rep and cap gene inhibitorysequences, allows expression and activation of the rep and cap proteinsand resulting in the replication and packaging of the AAV genome.

The cre protein may be provided in two alternative ways. The geneencoding the protein may be a separate component transfected into thedesired host cell. Alternatively, the host cell selected for expressionof the rAAV may express the cre protein constitutively or under aninducible promoter.

B. Triple Infection/Transfection Method

In one embodiment of the present invention, the method employs threevectors, i.e., recombinant viruses or plasmids, to infect/transfect aselected host cell for production of a rAAV. A first vector comprisesthe cre transgene operatively linked to expression control sequences. Asecond vector comprises the AAV rep and cap genes downstream of a spacersequence which is flanked by lox sites and which itself is downstream ofexpression control sequences. A third vector comprises the therapeuticminigene, i.e., a transgene flanked by AAV ITRs and regulatorysequences. Suitable techniques for introducing these vectors into thehost cell are discussed below and are known to those of skill in theart. When all vectors are present in a cell and the cell is providedwith helper functions, the rAAV is efficiently produced.

1. First Vector

As stated above, in a preferred embodiment, a first vector is arecombinant replication-defective adenovirus containing the cretransgene operatively linked to expression control sequences in the siteof adenovirus E1 deletion, e.g., Ad.CMV.NLS-CRE. See FIG. 5. Preferably,as in the examples below, the cre gene is operably linked to a suitablenuclear localization signal (NLS). A suitable NLS is a short sequence,i.e., in the range of about 21 bp, and may be readily synthesized usingconventional techniques, or engineered onto the vector by including theNLS sequences in a PCR primer. As described in detail in Example 1below, the cre gene and a nuclear localization signal (NLS) are obtainedfrom a previously described plasmid.

Desirably, the cre gene is under the control of a cytomegalovirus (CMV)immediate early promoter/enhancer [see, e.g., Boshart et al, Cell,41:521-530 (1985)]. However, other suitable promoters may be readilyselected by one of skill in the art. Useful promoters may beconstitutive promoters or regulated (inducible) promoters, which willenable control of the amount of the cre gene product to be expressed.For example, another suitable promoter includes, without limitation, theRous sarcoma virus LTR promoter/enhancer. Still other promoter/enhancersequences may be selected by one of skill in the art.

In addition, the recombinant virus also includes conventional regulatoryelements necessary to drive expression of the cre recombinase in a celltransfected with the vector. Such regulatory elements are known to thoseof skill in the art, including without limitation, polyA sequences,origins of replication, etc.

2. Second Vector

Another, “second”, vector useful in this embodiment of the method isdescribed in Example 2 as Ad.sp.Rep/Cap. It contains the AAV rep and capgenes downstream of a spacer sequence which is flanked by lox sites andwhich itself is downstream of expression control sequences.

The AAV rep and cap sequences are obtained by conventional means.Preferably, the promoter is the AAV P5 promoter. However, one of skillin the art may readily substitute other suitable promoters. Examples ofsuch promoters are discussed above in connection with the first vector.

The spacer is an intervening DNA sequence (STOP) between the promoterand the gene. It is flanked by loxP sites and contains multipletranslational start and stop codons. The spacer is designed to permituse of a “Recombination-Activated Gene Expression (RAGE)” strategy [B.Sauer, Methods Enzymol., 225:890-900 (1993)]. Such a strategy controlsthe expression of a given gene (in this case, rep/cap). The spacer mustbe excised by expression of the cre protein of the first vector and itsinteraction with the lox sequences to express rep/cap.

Currently, there are two particularly preferred spacers. These spacersinclude a 1600 bp DNA fragment containing the GFP cDNA, an intron and apolyadenylation signal (FIG. 1) which was derived from a commercialplasmid (Clontech) as described below. A second preferred spacer is a1300 bp fragment containing translational start and stop sequencesobtained as a 1.3 kbp ScaI-SmaI fragment of pBS64 as described [M. Antonand F. Graham, J. Virol., 69:4600-4606 (1995)]. Another desirable spaceris a 1000 bp fragment containing the neomycin resistance coding sequenceand a polyadenylation signal [Y. Kanegae et al, Nucl. Acids Res.,23:3816-3821 (1995)] (see, FIG. 2).

Using the information provided herein, one of skill in the art mayselect and design other suitable spacers, taking into consideration suchfactors as length, the presence of at least one set of translationalstart and stop signals, and optionally, the presence of polyadenylationsites. These spacers may contain genes, which typically incorporate thelatter two elements (i.e., the start/stop and polyA sites). Desirably,to reduce the possibility of recombination, the spacer is less than 2kbp in length. However, the invention is not so limited.

As stated above, the spacer is flanked by loxP sites, which arerecognized by the cre protein and participate in the deletion of thespacer. The sequences of loxP are publicly available from a variety ofsources [R. H. Hoess and K. Abremski, Proc. Natl. Acad. Sci., 81:1026-1029 (1984)]. Upon selection of a suitable spacer and making use ofknown techniques, one can readily engineer loxP sites onto the ends ofthe spacer sequence for use in the method of the invention.

In addition, the recombinant virus which carries the rep/cap genes andthe spacer, also includes conventional regulatory elements necessary todrive expression of rep and cap in a cell transfected with therecombinant virus, following excision of the loxP-flanked spacer by thecre recombinase. Such regulatory elements are known to those of skill inthe art.

3. Third Vector

The third vector contains a minigene, which is defined as a sequencewhich comprises a suitable transgene, a promoter, and other regulatoryelements necessary for expression of the transgene, all flanked by AAVITRs. In the examples below, where the third vector carries the LacZgene, the presence of rAAV is detected by assays for beta-galactosidaseactivity. However, desirably, the third vector carries a therapeuticgene which can be delivered to an animal via the rAAV produced by thismethod.

The AAV sequences employed are preferably the cis-acting 5′ and 3′inverted terminal repeat (ITR) sequences [See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp.155-168(1990)]. The ITR sequences are about 143 bp in length. Preferably,substantially the entire sequences encoding the ITRs are used in thevectors, although some degree of minor modification of these sequencesis expected to be permissible for this use. The ability to modify theseITR sequences is within the skill of the art. [See, e.g., texts such asSambrook et al, “Molecular Cloning. A Laboratory Manual.”, 2d edit.,Cold Spring Harbor Laboratory, New York (1989); Carter et al, citedabove; and K. Fisher et al., J. Virol., 70:520-532 (1996)].

The AAV ITR sequences may be obtained from any known AAV, includingpresently identified human AAV types. Similarly, AAVs known to infectother animals may also be employed in the vector constructs of thisinvention. The selection of the AAV is not anticipated to limit thefollowing invention. A variety of AAV strains, types 1-4, are availablefrom the American Type Culture Collection or available by request from avariety of commercial and institutional sources. In the followingexemplary embodiment an AAV-2 is used for convenience.

The 5′ and 3′ AAV ITR sequences flank the selected transgene sequenceand associated regulatory elements. The transgene sequence of the vectoris a nucleic acid sequence heterologous to the AAV sequence, whichencodes a polypeptide or protein of interest. The composition of thetransgene sequence will depend upon the use to which the resultingvector will be put. For example, one type of transgene sequence includesa reporter sequence, which upon expression produces a detectable signal.Such reporter sequences include without limitation an E. colibeta-galactosidase (LacZ) cDNA, an alkaline phosphatase gene and a greenfluorescent protein gene. These sequences, when associated withregulatory elements which drive their expression, provide signalsdetectable by conventional means, e.g., ultraviolet wavelengthabsorbance, visible color change, etc.

A more preferred type of transgene sequence is a therapeutic gene whichexpresses a desired gene product in a host cell. These therapeuticnucleic acid sequences typically encode products for administration andexpression in a patient in vivo or ex vivo to replace or correct aninherited or non-inherited genetic defect or treat an epigeneticdisorder or disease. The selection of the transgene sequence is not alimitation of this invention.

In addition to the major elements identified above, the minigene alsoincludes conventional regulatory elements necessary to drive expressionof the transgene in a cell transfected with this vector. Thus, theminigene comprises a selected promoter which is linked to the transgeneand located within the transgene between the AAV ITR sequences.

Selection of the promoter used to drive expression of the transgene is aroutine matter and is not a limitation of the vector. Useful promotersinclude those which are discussed above in connection with the firstvector component.

The minigene also desirably contains heterologous nucleic acid sequencesincluding sequences providing signals required for efficientpolyadenylation of the transcript and introns with functional splicedonor and acceptor sites. A common poly-A sequence which is employed inthe exemplary vectors of this invention is that derived from thepapovavirus SV-40. The poly-A sequence generally is inserted followingthe transgene sequences and before the 3′ AAV ITR sequence. A commonintron sequence is also derived from SV-40, and is referred to as theSV-40 T intron sequence. A minigene of the present invention may alsocontain such an intron, desirably located between the promoter/enhancersequence and the transgene. Selection of these and other common vectorelements are conventional and many such sequences are available [see,e.g., Sambrook et al, and references cited therein].

The rAAV vector containing the minigene may be carried on a plasmidbackbone and used to transfect a selected host cell or may be flanked byviral sequences (e.g., adenoviral sequences) which permit it to infectthe selected host cell. Suitable Ad/AAV recombinant viruses may beproduced in accordance with known techniques. See, e.g., Internationalpatent applications WO96/13598, published May 9, 1996; WO95/23867published Sep. 8, 1995, and WO95/06743 published Mar. 9, 1995, which areincorporated by reference herein.

C. Host Cell/Double Infection or Transfection System

In another embodiment of the method of this invention, a packaging cellline is constructed which expresses the cre recombinase. According tothis aspect of the method, this cell line expressing the cre recombinasecan be substituted for the vector or plasmid bearing the cre gene, asdescribed above. Thus, only the second and third vectors described aboveare subsequently introduced into the cell.

An exemplary suitable cre expressing cell line has been generated usingthe vector illustrated in FIG. 3. Generation of this cell line isdescribed in detail in Example 4 below. However, the present inventionis not limited to these constructs. Given the information providedherein, one of skill in the art can readily generate another plasmidcontaining a suitable selectable marker (e.g., neo^(R)). Such a plasmidmay then be used for the generation of a cre recombinase-expressing cellline according to the invention.

Having obtained such a cre-expressing cell line, this cell line can beinfected (or transfected) with the vector containing the rep/cap genesand the vector containing the minigene described above.

D. Production of Vectors and rAAV

Assembly of the selected DNA sequences contained within each of thevectors described above utilize conventional techniques. Such techniquesinclude cDNA cloning such as those described in texts [Sambrook et al,cited above], use of overlapping oligonucleotide sequences of theadenovirus, AAV genome combined with polymerase chain reaction, and anyother suitable methods which provide the desired nucleotide sequence.

Whether using the three vector system, or the cre-expressing host celland two vectors, introduction of the vectors into the host cell isaccomplished using known techniques. Where appropriate, standardtransfection and co-transfection techniques are employed, e.g., CaPO₄transfection techniques using the complementation human embryonic kidney(HEK) 293 cell line (a human kidney cell line containing a functionaladenovirus E1a gene which provides a transacting E1a protein). Otherconventional methods employed in this invention include homologousrecombination of the viral genomes, plaquing of viruses in agar overlay,methods of measuring signal generation, and the like.

Following infection/transfection, the host cell is then cultured understandard conditions, to enable production of the rAAV. See, e.g., F. L.Graham and L. Prevec, Methods Mol. Biol., 7:109-128 (1991). Desirably,once the rAAV is identified by conventional means, it may be recoveredusing standard techniques and purified.

The following examples illustrate the preferred methods of theinvention. These examples are illustrative only and are not intended tolimit the scope of the invention.

EXAMPLE 1 Construction of Ad.CMV.NLS-CRE

The construction of a recombinant adenovirus containing a nuclearlocalization signal and the cre gene under control of a cytomegaloviruspromoter is described below, with reference to FIG. 5.

The nls-Cre cDNA was isolated from the plasmid pexCANCRE [Y. Kanegae etal, Nucl. Acids Res., 23:3816-3821 (1995)] by digestion with Sfcil andPacd and then blunt ended with Klenow and T4 DNA polymerase. The NLS-Crefragment was then cloned into the EcoRV site of the plasmid pAd.CMV.Link(a plasmid containing the human Ad5 sequences, map units 0 to 16, whichis deleted of E1a and E1b as described in X. Ye et al, J. Biol. Chem.,271:3639-3646 (1996). The orientation and presence of the nuclearlocalization signal in the resulting plasmid pAd.CMV.NLS-CRE wasverified by sequencing.

To produce the recombinant adenovirus carrying the cre transgene, thepAd.CMV.NLS-CRE recombinant vector was co-transfected with the Ad dl327backbone into 293 cells. Ten days later, 15 plaques were picked up and 5of them were expanded on 293 cells. Viruses were screened for theirrecombinase activity by assessing their ability to remove a spacerpositioned between the CAG promoter (beta-actin) and the bacterial LacZcoding sequence using an adenoviral construct described in Y. Kanegae etal, Nucl. Acids Res., 23:3816-3821 (1995). Two viruses tested positivefor beta-galactosidase activity, indicating cre recombinase activity. Asdesired, these recombinant viruses may be purified by two rounds ofplaque purification.

EXAMPLE 2 Construction of Ad.sp.Rep/Cap

An exemplary recombinant adenovirus containing the AAV rep and cap genesmay be produced as follows.

An AAV P5 promoter was obtained from the 121 bp XbaI-BamHI fragment fromplasmid psub201, which contains the entire AAV2 genome [R. J. Samulskiet al, J. Virol., 61:3096-3101 (1987)] by PCR using the following primerpairs:

XbaI ITR rightward: SEQ ID NO:2:

GGCCTCTAGATGGAGGGGTGGAGTCGTGAC;

BamP5 rightward: SEQ ID NO:3:

GGCCGGATCCAACGCGCAGCCGCCATGCCG;

Bam P5 leftward: SEQ ID NO:4:

GGCCGGATCCCAAACCTCCCGCTTCAAAAT;

SacI leftward: SEQ ID NO:5:

GGCCGAGCTCAGGCTGGGTTTTGGGGAGCA.

A 5′ portion of the Rep/Cap gene was similarly excised via PCR from aBamI-SacI fragment (504 bp) obtained from psub201. The BamHI PCR primercreates a unique site between the rep mRNA and the first rep ATG. The P5promoter and the Rep/Cap gene fragment were subcloned into the XbaI-SacIsites of the pSP72 vector (Promega), resulting in P5.Rep/Cap. The spacerDNA, a 1300 bp fragment flanked by loxP sites, was obtained from theplasmid pMA19 [M. Anton and F. Graham, J. Virol., 69:4600-4606 (1995)]following digestion with BamHI. This spacer DNA was cloned into theunique BamHI site of the P5.Rep/Cap construct, resulting in theP5.Spacer.Rep/Cap construct.

The complete fragment containing the P5 promoter, the spacer and therep/cap genes was obtained by subcloning the 3′ portion of the Rep/Capgene (SacI/blunt ended fragment, 3680 bp) into the SacI-EcoRV sites ofthe P5.Spacer.Rep/Cap plasmid. The 3′ portion of the Rep/cap gene wasisolated from the SSV9 plasmid (which contains a complete wild-type AAVgenome) as a SacI-blunt ended fragment. This involved digesting SSV9with XbaI, filling the XbaI site with Klenow and liberating the fragmentby digesting with SacI.

The complete fragment containing the P5 promoter, the spacer and therep/cap sequence was subcloned into the BglII site of the pAd.linkvector. This was accomplished by adding a BglII linker at the 5′ end ofthe P5.Spacer.Rep/Cap plasmid construct and using the BglII site locatedat the 3′ end of the multiple cloning site of pSP72.

The resulting plasmid (11250 bp) contains Ad5 map units (mu) 0-1, the P5promoter, the spacer sequence flanked by loxP sites, rep/cap, and Ad5 mu9-16. This plasmid is termed pAd.P5.spacer.Rep/Cap [FIG. 4].

To produce recombinant adenovirus capable of expressing rep and cap,pAd.P5.spacer.Rep/Cap was first used to transform a cre-expressingbacterial strain E. coli strain BNN132 (ATCC Accession No. 47059) inorder to determine whether the spacer could be removed afterrecombination between the loxP sites (catalyzed by the cre recombinase).Analysis on agarose gels of the plasmid DNA isolated from severaltransformed colonies showed that, indeed, most of the constructsanalyzed lost the spacer following transformation (data not shown).

The plasmid P5.spacer.Rep/Cap was also co-transfected with the Ad dl327backbone in HEK 293 cells. Ten days later, 20 plaques were picked up andexpanded. The structure of the viruses was analyzed by Southern blotusing the complete AAV genome and the 1300 bp DNA spacer as probes. Oneplaque (P3) showed the expected band pattern after digestion with therestriction enzyme BamHI (data not shown).

Similar constructs may be made using other suitable spacers. Forexample, a 1600 bp spacer was derived from plasmid phGFP-S65T plasmid(Clontech) which contains the humanized GFP gene. phGFP-S65T was cutwith the restriction enzymes HindIIUI and BamHI. After adding a BglIIlinker at the 5′ end (BglII is compatible with BamHI), the 1.6 kbfragment was subdloned into the BamIH site of the flox vector [H. Gu etal, Science, 265:103-106 (1994)] in order to add a loxP site on eachside of the fragment. The GFP DNA fragment flanked by loxP sites wassubsequently cut with PvuI and SmaI and subdloned into the EcoRV site ofthe Bluescript II cloning vector (Stratagene). The resulting GFP spacercan be used to construct a P5.spacer.Rep/cap plasmid or adenovirus asdescribed above.

EXAMPLE 3 Production of rAAV

The supernatant from several plaques (containing viruses) obtained fromthe study described in Example 2 was tested for the ability to produceAAV in a functional assay involving the adenovirus encoding the creprotein constructed as described in Example 1 above and pAV.CMVLacZ.

The plasmid AV.CMVLacZ is a rAAV cassette in which rep and cap genes arereplaced with a minigene expressing β-galactosidase from a CMV promoter.The linear arrangement of AV.CMVLacZ includes:

(a) the 5′ AAV ITR (bp 1-173) obtained by PCR using pAV2 [C. A. Laughlinet al, Gene, 23: 65-73 (1983)] as template [nucleotide numbers 365-538of SEQ ID NO:1];

(b) a CMV immediate early enhancer/promoter [Boshart et al, Cell,41:521-530 (1985); nucleotide numbers 563-1157 of SEQ ID NO: 1],

(c) an SV40 intron (nucleotide numbers 1178-1179 of SEQ ID NO: 1),

(d) E. coli beta-galactosidase cDNA (nucleotide numbers 1356-4827 of SEQID NO: 1),

(e) an SV40 polyadenylation signal (a 237 BamHI-BclI restrictionfragment containing the cleavage/poly-A signals from both the early andlate transcription units; nucleotide numbers 4839-5037 of SEQ ID NO:1)and

(f) 3′AAV ITR, obtained from pAV2 as a SnaBI-BglII fragment (nucleotidenumbers 5053-5221 of SEQ ID NO:1).

The functional assay was performed by infecting 293 cells with the crevirus and the Rep/Cap virus (multiplicity of infection (MOI) 10)followed by a transfection 2 hours later with 5 μg pAV.CMVLacZ.Forty-eight hours later, cells were harvested and freeze-thawed.One-fifth of the supernatant (containing rAAV) was used to infect 293cells. Twenty-four hours later an X-gal assay was performed.

Viruses from plaque #3 yielded positive for beta-galactosidasetransduction in this assay. Supernatant from plaque #3 was used in asecond round of purification (plaque amplification). Twenty plaques werepicked up and expanded.

EXAMPLE 4 Production of Cre Expressing Cell Line

A plasmid vector, pG.CMV.nls.cre was constructed as follows for use intransfecting 293 cells. The nls-Cre cDNA was isolated from the plasmidpexCANCRE (Kanegae, cited above) as described in Example 1 above. Thenls-Cre fragment was then subcloned into the XbaI sites of vector pGdownstream of a CMV promoter. This plasmid vector is illustrated in FIG.3 and contains a human growth hormone (hGH) termination sequence, anSV40 ori signal, a neomycin resistance marker, an SV40 polyadenylationsite, an ampicillin marker, on a backbone of pUC19.

This plasmid was transfected into 293 cells using conventionaltechniques. Cells were selected in the presence of G-418 for neomycinresistance. Cells were identified by infecting them at different MOI (1to 100) with Ad.CAG.Sp.LacZ, an adenovirus containing the bacterial LacZcoding sequence separated from its beta-actin (CAG) promoter by aneomycin spacer DNA flanked by two loxP sites followed by the bacterialLacZ gene. Cells were selected on the basis of their ability to removethe spacer fragment inducing the expression of the LacZ gene. AfterX-gal staining, six cell lines were found to be positive. DNA from theseinfected cells was isolated and analyzed by Southern blot using thespacer DNA (NEO) as a probe. Results shown in FIG. 6A, with reference toTable 1, and FIGS. 6B-6D indicate that cell line #2 can remove the DNAspacer with much more efficacy than the other 293/cre cell linesanalyzed.

TABLE 1 NEO Probe Without Recombination With Recombination 6200 62005200

EXAMPLE 5 Generation of the Ad.GFP Rep/Cap

As described in Example 2 for the construction of the Ad.Sp.Rep/Capvirus, the link plasmid containing the P5 promoter, the GFP spacerflanked by two loxP sites and the Rep and Cap coding sequences wasco-transfected with the Ad dl327 backbone into HEK 293 cells. Ten dayslater, 20 plaques were picked up and expanded. During this expansion,the monolayer of HEK 293 cells were screened for the expression of GFPby microscopic analysis using a mercury lamp with a 470-490 nm band-passexcitation filter (Nikon). One of the monolayers (from plaque #13)showed a region positive for the expression of GFP. This region wasfurther expanded and purified by two other rounds of plaquepurification. The presence of the Ad.GFP.Rep/Cap virus was monitored bythe expression of GFP, as described, and/or by the expression of the Repand Cap proteins by Western blot analysis using specific monoclonalantibodies (American Research Products, Inc.). One cell lysate (from onepurified plaque) containing the Ad.GFP rep/cap was used in order toinfect 293 cells (adenovirus preparation with 40×150 mm dishes of HEK293 cells). A total of 6.86×10¹³ particles/ml were obtained afterpurification. This virus is currently being tested for the production ofrAAV, as described in Example 3.

EXAMPLE 6 Construction of the Ad.TRE.CMV.GFP.Rep/Cap

FIG. 7 shows the final structure of the Ad.TRE.CMV.Rep/Cap virus. TheAAV P5 promoter was replaced by the tetracycline (Tet) induciblepromoter (Clontech). This promoter contains the tetracycline responsiveelements (TRE) followed by the CMV minimal promoter without the CMVenhancer. This promoter is inducible in the presence of the antibioticdoxycycline (Sigma) in the 293/Tet-On cell line (Clontech) whichcontains a stable gene expressing the rTetR (reverse Tet repressor)fused to the GP16 transcriptional activation domain. The objective hereis to construct a double inducible expression system in order to limitthe expression of the cytotoxic Rep gene products. In order to fullyinduce the expression of the Rep and Cap genes, the virus must be in thepresence of 1—the cre recombinase (in order to delete the GFP spacer asdescribed previously) and 2—the Tet-On inducible factor doxycycline(DOX).

The link plasmid containing the construct described above was used totransfect HEK 293 cells in the presence or the absence of DOX and/or thecre recombinase (from the adenovirus expressing nls-cre). Proteins fromcell homogenates were analyzed by Western blot using the Rep antibodies.Rep proteins are fully induced only in the presence of DOX and the crerecombinase.

In order to construct pAd.TRE.CMV.link. 1, the pTRE plasmid (Clontech)was cut with the restriction endonucleases Xho and EcoR1 to isolate theTRE and the minimal CMV promoter. The Xho and EcoR1 sites were filledwith Klenow and the 448 bp fragment was inserted into the EcoRV site ofthe pAdlink. 1 plasmid. The GFP.Rep/Cap fragment was subsequently cutwith ClaI and BglIII and inserted into the pAd.TRE.CMV.link. 1 cut withClaI and BamHI.

This link recombinant plasmid was co-transfected with the Ad dl327backbone in HEK 293 cells. Ten days later, 20 plaques were picked up andexpanded. These plaques are currently being analyzed for the expressionof GFP and the Rep and Cap proteins. Two adenoviruses expressing largeamounts of rep proteins were identified. These viruses are currentlybeing purified and studied.

Numerous modifications and variations of the present invention areincluded in the above-identified specification and are expected to beobvious to one of skill in the art. Such modifications and alterationsto the processes of the present invention are believed to be encompassedin the scope of the claims appended hereto.

5 10398 base pairs nucleic acid double unknown cDNA 1 GAATTCGCTAGCATCATCAA TAATATACCT TATTTTGGAT TGAAGCCAAT ATGATAATGA 60 GGGGGTGGAGTTTGTGACGT GGCGCGGGGC GTGGGAACGG GGCGGGTGAC GTAGTAGTGT 120 GGCGGAAGTGTGATGTTGCA AGTGTGGCGG AACACATGTA AGCGACGGAT GTGGCAAAAG 180 TGACGTTTTTGGTGTGCGCC GGTGTACACA GGAAGTGACA ATTTTCGCGC GGTTTTAGGC 240 GGATGTTGTAGTAAATTTGG GCGTAACCGA GTAAGATTTG GCCATTTTCG CGGGAAAACT 300 GAATAAGAGGAAGTGAAATC TGAATAATTT TGTGTTACTC ATAGCGCGTA ATATTTGTCT 360 AGGGAGATCTGCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC 420 GGGCGACCTTTGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA 480 ACTCCATCACTAGGGGTTCC TTGTAGTTAA TGATTAACCC GCCATGCTAC TTATCTACAA 540 TTCGAGCTTGCATGCCTGCA GGTCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA 600 CCGCCCAACGACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA 660 ATAGGGACTTTCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC CCACTTGGCA 720 GTACATCAAGTGTATCATAT GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG 780 CCCGCCTGGCATTATGCCCA GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC 840 TACGTATTAGTCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT 900 GGATAGCGGTTTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT 960 TTGTTTTGGCACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG 1020 ACGCAAATGGGCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC TCGTTTAGTG 1080 AACCGTCAGATCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG AAGACACCGG 1140 GACCGATCCAGCCTCCGGAC TCTAGAGGAT CCGGTACTCG AGGAACTGAA AAACCAGAAA 1200 GTTAACTGGTAAGTTTAGTC TTTTTGTCTT TTATTTCAGG TCCCGGATCC GGTGGTGGTG 1260 CAAATCAAAGAACTGCTCCT CAGTGGATGT TGCCTTTACT TCTAGGCCTG TACGGAAGTG 1320 TTACTTCTGCTCTAAAAGCT GCGGAATTGT ACCCGCGGCC GCAATTCCCG GGGATCGAAA 1380 GAGCCTGCTAAAGCAAAAAA GAAGTCACCA TGTCGTTTAC TTTGACCAAC AAGAACGTGA 1440 TTTTCGTTGCCGGTCTGGGA GGCATTGGTC TGGACACCAG CAAGGAGCTG CTCAAGCGCG 1500 ATCCCGTCGTTTTACAACGT CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC 1560 TTGCAGCACATCCCCCTTTC GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC 1620 CTTCCCAACAGTTGCGCAGC CTGAATGGCG AATGGCGCTT TGCCTGGTTT CCGGCACCAG 1680 AAGCGGTGCCGGAAAGCTGG CTGGAGTGCG ATCTTCCTGA GGCCGATACT GTCGTCGTCC 1740 CCTCAAACTGGCAGATGCAC GGTTACGATG CGCCCATCTA CACCAACGTA ACCTATCCCA 1800 TTACGGTCAATCCGCCGTTT GTTCCCACGG AGAATCCGAC GGGTTGTTAC TCGCTCACAT 1860 TTAATGTTGATGAAAGCTGG CTACAGGAAG GCCAGACGCG AATTATTTTT GATGGCGTTA 1920 ACTCGGCGTTTCATCTGTGG TGCAACGGGC GCTGGGTCGG TTACGGCCAG GACAGTCGTT 1980 TGCCGTCTGAATTTGACCTG AGCGCATTTT TACGCGCCGG AGAAAACCGC CTCGCGGTGA 2040 TGGTGCTGCGTTGGAGTGAC GGCAGTTATC TGGAAGATCA GGATATGTGG CGGATGAGCG 2100 GCATTTTCCGTGACGTCTCG TTGCTGCATA AACCGACTAC ACAAATCAGC GATTTCCATG 2160 TTGCCACTCGCTTTAATGAT GATTTCAGCC GCGCTGTACT GGAGGCTGAA GTTCAGATGT 2220 GCGGCGAGTTGCGTGACTAC CTACGGGTAA CAGTTTCTTT ATGGCAGGGT GAAACGCAGG 2280 TCGCCAGCGGCACCGCGCCT TTCGGCGGTG AAATTATCGA TGAGCGTGGT GGTTATGCCG 2340 ATCGCGTCACACTACGTCTG AACGTCGAAA ACCCGAAACT GTGGAGCGCC GAAATCCCGA 2400 ATCTCTATCGTGCGGTGGTT GAACTGCACA CCGCCGACGG CACGCTGATT GAAGCAGAAG 2460 CCTGCGATGTCGGTTTCCGC GAGGTGCGGA TTGAAAATGG TCTGCTGCTG CTGAACGGCA 2520 AGCCGTTGCTGATTCGAGGC GTTAACCGTC ACGAGCATCA TCCTCTGCAT GGTCAGGTCA 2580 TGGATGAGCAGACGATGGTG CAGGATATCC TGCTGATGAA GCAGAACAAC TTTAACGCCG 2640 TGCGCTGTTCGCATTATCCG AACCATCCGC TGTGGTACAC GCTGTGCGAC CGCTACGGCC 2700 TGTATGTGGTGGATGAAGCC AATATTGAAA CCCACGGCAT GGTGCCAATG AATCGTCTGA 2760 CCGATGATCCGCGCTGGCTA CCGGCGATGA GCGAACGCGT AACGCGAATG GTGCAGCGCG 2820 ATCGTAATCACCCGAGTGTG ATCATCTGGT CGCTGGGGAA TGAATCAGGC CACGGCGCTA 2880 ATCACGACGCGCTGTATCGC TGGATCAAAT CTGTCGATCC TTCCCGCCCG GTGCAGTATG 2940 AAGGCGGCGGAGCCGACACC ACGGCCACCG ATATTATTTG CCCGATGTAC GCGCGCGTGG 3000 ATGAAGACCAGCCCTTCCCG GCTGTGCCGA AATGGTCCAT CAAAAAATGG CTTTCGCTAC 3060 CTGGAGAGACGCGCCCGCTG ATCCTTTGCG AATACGCCCA CGCGATGGGT AACAGTCTTG 3120 GCGGTTTCGCTAAATACTGG CAGGCGTTTC GTCAGTATCC CCGTTTACAG GGCGGCTTCG 3180 TCTGGGACTGGGTGGATCAG TCGCTGATTA AATATGATGA AAACGGCAAC CCGTGGTCGG 3240 CTTACGGCGGTGATTTTGGC GATACGCCGA ACGATCGCCA GTTCTGTATG AACGGTCTGG 3300 TCTTTGCCGACCGCACGCCG CATCCAGCGC TGACGGAAGC AAAACACCAG CAGCAGTTTT 3360 TCCAGTTCCGTTTATCCGGG CAAACCATCG AAGTGACCAG CGAATACCTG TTCCGTCATA 3420 GCGATAACGAGCTCCTGCAC TGGATGGTGG CGCTGGATGG TAAGCCGCTG GCAAGCGGTG 3480 AAGTGCCTCTGGATGTCGCT CCACAAGGTA AACAGTTGAT TGAACTGCCT GAACTACCGC 3540 AGCCGGAGAGCGCCGGGCAA CTCTGGCTCA CAGTACGCGT AGTGCAACCG AACGCGACCG 3600 CATGGTCAGAAGCCGGGCAC ATCAGCGCCT GGCAGCAGTG GCGTCTGGCG GAAAACCTCA 3660 GTGTGACGCTCCCCGCCGCG TCCCACGCCA TCCCGCATCT GACCACCAGC GAAATGGATT 3720 TTTGCATCGAGCTGGGTAAT AAGCGTTGGC AATTTAACCG CCAGTCAGGC TTTCTTTCAC 3780 AGATGTGGATTGGCGATAAA AAACAACTGC TGACGCCGCT GCGCGATCAG TTCACCCGTG 3840 CACCGCTGGATAACGACATT GGCGTAAGTG AAGCGACCCG CATTGACCCT AACGCCTGGG 3900 TCGAACGCTGGAAGGCGGCG GGCCATTACC AGGCCGAAGC AGCGTTGTTG CAGTGCACGG 3960 CAGATACACTTGCTGATGCG GTGCTGATTA CGACCGCTCA CGCGTGGCAG CATCAGGGGA 4020 AAACCTTATTTATCAGCCGG AAAACCTACC GGATTGATGG TAGTGGTCAA ATGGCGATTA 4080 CCGTTGATGTTGAAGTGGCG AGCGATACAC CGCATCCGGC GCGGATTGGC CTGAACTGCC 4140 AGCTGGCGCAGGTAGCAGAG CGGGTAAACT GGCTCGGATT AGGGCCGCAA GAAAACTATC 4200 CCGACCGCCTTACTGCCGCC TGTTTTGACC GCTGGGATCT GCCATTGTCA GACATGTATA 4260 CCCCGTACGTCTTCCCGAGC GAAAACGGTC TGCGCTGCGG GACGCGCGAA TTGAATTATG 4320 GCCCACACCAGTGGCGCGGC GACTTCCAGT TCAACATCAG CCGCTACAGT CAACAGCAAC 4380 TGATGGAAACCAGCCATCGC CATCTGCTGC ACGCGGAAGA AGGCACATGG CTGAATATCG 4440 ACGGTTTCCATATGGGGATT GGTGGCGACG ACTCCTGGAG CCCGTCAGTA TCGGCGGAAT 4500 TACAGCTGAGCGCCGGTCGC TACCATTACC AGTTGGTCTG GTGTCAAAAA TAATAATAAC 4560 CGGGCAGGCCATGTCTGCCC GTATTTCGCG TAAGGAAATC CATTATGTAC TATTTAAAAA 4620 ACACAAACTTTTGGATGTTC GGTTTATTCT TTTTCTTTTA CTTTTTTATC ATGGGAGCCT 4680 ACTTCCCGTTTTTCCCGATT TGGCTACATG ACATCAACCA TATCAGCAAA AGTGATACGG 4740 GTATTATTTTTGCCGCTATT TCTCTGTTCT CGCTATTATT CCAACCGCTG TTTGGTCTGC 4800 TTTCTGACAAACTCGGCCTC GACTCTAGGC GGCCGCGGGG ATCCAGACAT GATAAGATAC 4860 ATTGATGAGTTTGGACAAAC CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA 4920 ATTTGTGATGCTATTGCTTT ATTTGTAACC ATTATAAGCT GCAATAAACA AGTTAACAAC 4980 AACAATTGCATTCATTTTAT GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTCGGAT 5040 CCTCTAGAGTCGAGTAGATA AGTAGCATGG CGGGTTAATC ATTAACTACA AGGAACCCCT 5100 AGTGATGGAGTTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC 5160 AAAGGTCGCCCGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC GAGCGCGCAG 5220 CAGATCTGGAAGGTGCTGAG GTACGATGAG ACCCGCACCA GGTGCAGACC CTGCGAGTGT 5280 GGCGGTAAACATATTAGGAA CCAGCCTGTG ATGCTGGATG TGACCGAGGA GCTGAGGCCC 5340 GATCACTTGGTGCTGGCCTG CACCCGCGCT GAGTTTGGCT CTAGCGATGA AGATACAGAT 5400 TGAGGTACTGAAATGTGTGG GCGTGGCTTA AGGGTGGGAA AGAATATATA AGGTGGGGGT 5460 CTTATGTAGTTTTGTATCTG TTTTGCAGCA GCCGCCGCCG CCATGAGCAC CAACTCGTTT 5520 GATGGAAGCATTGTGAGCTC ATATTTGACA ACGCGCATGC CCCCATGGGC CGGGGTGCGT 5580 CAGAATGTGATGGGCTCCAG CATTGATGGT CGCCCCGTCC TGCCCGCAAA CTCTACTACC 5640 TTGACCTACGAGACCGTGTC TGGAACGCCG TTGGAGACTG CAGCCTCCGC CGCCGCTTCA 5700 GCCGCTGCAGCCACCGCCCG CGGGATTGTG ACTGACTTTG CTTTCCTGAG CCCGCTTGCA 5760 AGCAGTGCAGCTTCCCGTTC ATCCGCCCGC GATGACAAGT TGACGGCTCT TTTGGCACAA 5820 TTGGATTCTTTGACCCGGGA ACTTAATGTC GTTTCTCAGC AGCTGTTGGA TCTGCGCCAG 5880 CAGGTTTCTGCCCTGAAGGC TTCCTCCCCT CCCAATGCGG TTTAAAACAT AAATAAAAAA 5940 CCAGACTCTGTTTGGATTTG GATCAAGCAA GTGTCTTGCT GTCTTTATTT AGGGGTTTTG 6000 CGCGCGCGGTAGGCCCGGGA CCAGCGGTCT CGGTCGTTGA GGGTCCTGTG TATTTTTTCC 6060 AGGACGTGGTAAAGGTGACT CTGGATGTTC AGATACATGG GCATAAGCCC GTCTCTGGGG 6120 TGGAGGTAGCACCACTGCAG AGCTTCATGC TGCGGGGTGG TGTTGTAGAT GATCCAGTCG 6180 TAGCAGGAGCGCTGGGCGTG GTGCCTAAAA ATGTCTTTCA GTAGCAAGCT GATTGCCAGG 6240 GGCAGGCCCTTGGTGTAAGT GTTTACAAAG CGGTTAAGCT GGGATGGGTG CATACGTGGG 6300 GATATGAGATGCATCTTGGA CTGTATTTTT AGGTTGGCTA TGTTCCCAGC CATATCCCTC 6360 CGGGGATTCATGTTGTGCAG AACCACCAGC ACAGTGTATC CGGTGCACTT GGGAAATTTG 6420 TCATGTAGCTTAGAAGGAAA TGCGTGGAAG AACTTGGAGA CGCCCTTGTG ACCTCCAAGA 6480 TTTTCCATGCATTCGTCCAT AATGATGGCA ATGGGCCCAC GGGCGGCGGC CTGGGCGAAG 6540 ATATTTCTGGGATCACTAAC GTCATAGTTG TGTTCCAGGA TGAGATCGTC ATAGGCCATT 6600 TTTACAAAGCGCGGGCGGAG GGTGCCAGAC TGCGGTATAA TGGTTCCATC CGGCCCAGGG 6660 GCGTAGTTACCCTCACAGAT TTGCATTTCC CACGCTTTGA GTTCAGATGG GGGGATCATG 6720 TCTACCTGCGGGGCGATGAA GAAAACGGTT TCCGGGGTAG GGGAGATCAG CTGGGAAGAA 6780 AGCAGGTTCCTGAGCAGCTG CGACTTACCG CAGCCGGTGG GCCCGTAAAT CACACCTATT 6840 ACCGGGTGCAACTGGTAGTT AAGAGAGCTG CAGCTGCCGT CATCCCTGAG CAGGGGGGCC 6900 ACTTCGTTAAGCATGTCCCT GACTCGCATG TTTTCCCTGA CCAAATCCGC CAGAAGGCGC 6960 TCGCCGCCCAGCGATAGCAG TTCTTGCAAG GAAGCAAAGT TTTTCAACGG TTTGAGACCG 7020 TCCGCCGTAGGCATGCTTTT GAGCGTTTGA CCAAGCAGTT CCAGGCGGTC CCACAGCTCG 7080 GTCACCTGCTCTACGGCATC TCGATCCAGC ATATCTCCTC GTTTCGCGGG TTGGGGCGGC 7140 TTTCGCTGTACGGCAGTAGT CGGTGCTCGT CCAGACGGGC CAGGGTCATG TCTTTCCACG 7200 GGCGCAGGGTCCTCGTCAGC GTAGTCTGGG TCACGGTGAA GGGGTGCGCT CCGGGCTGCG 7260 CGCTGGCCAGGGTGCGCTTG AGGCTGGTCC TGCTGGTGCT GAAGCGCTGC CGGTCTTCGC 7320 CCTGCGCGTCGGCCAGGTAG CATTTGACCA TGGTGTCATA GTCCAGCCCC TCCGCGGCGT 7380 GGCCCTTGGCGCGCAGCTTG CCCTTGGAGG AGGCGCCGCA CGAGGGGCAG TGCAGACTTT 7440 TGAGGGCGTAGAGCTTGGGC GCGAGAAATA CCGATTCCGG GGAGTAGGCA TCCGCGCCGC 7500 AGGCCCCGCAGACGGTCTCG CATTCCACGA GCCAGGTGAG CTCTGGCCGT TCGGGGTCAA 7560 AAACCAGGTTTCCCCCATGC TTTTTGATGC GTTTCTTACC TCTGGTTTCC ATGAGCCGGT 7620 GTCCACGCTCGGTGACGAAA AGGCTGTCCG TGTCCCCGTA TACAGACTTG AGAGGCCTGT 7680 CCTCGACCGATGCCCTTGAG AGCCTTCAAC CCAGTCAGCT CCTTCCGGTG GGCGCGGGGC 7740 ATGACTATCGTCGCCGCACT TATGACTGTC TTCTTTATCA TGCAACTCGT AGGACAGGTG 7800 CCGGCAGCGCTCTGGGTCAT TTTCGGCGAG GACCGCTTTC GCTGGAGCGC GACGATGATC 7860 GGCCTGTCGCTTGCGGTATT CGGAATCTTG CACGCCCTCG CTCAAGCCTT CGTCACTGGT 7920 CCCGCCACCAAACGTTTCGG CGAGAAGCAG GCCATTATCG CCGGCATGGC GGCCGACGCG 7980 CTGGGCTACGTCTTGCTGGC GTTCGCGACG CGAGGCTGGA TGGCCTTCCC CATTATGATT 8040 CTTCTCGCTTCCGGCGGCAT CGGGATGCCC GCGTTGCAGG CCATGCTGTC CAGGCAGGTA 8100 GATGACGACCATCAGGGACA GCTTCAAGGA TCGCTCGCGG CTCTTACCAG CCTAACTTCG 8160 ATCACTGGACCGCTGATCGT CACGGCGATT TATGCCGCCT CGGCGAGCAC ATGGAACGGG 8220 TTGGCATGGATTGTAGGCGC CGCCCTATAC CTTGTCTGCC TCCCCGCGTT GCGTCGCGGT 8280 GCATGGAGCCGGGCCACCTC GACCTGAATG GAAGCCGGCG GCACCTCGCT AACGGATTCA 8340 CCACTCCAAGAATTGGAGCC AATCAATTCT TGCGGAGAAC TGTGAATGCG CAAACCAACC 8400 CTTGGCAGAACATATCCATC GCGTCCGCCA TCTCCAGCAG CCGCACGCGG CGCATCTCGG 8460 GCAGCGTTGGGTCCTGGCCA CGGGTGCGCA TGATCGTGCT CCTGTCGTTG AGGACCCGGC 8520 TAGGCTGGCGGGGTTGCCTT ACTGGTTAGC AGAATGAATC ACCGATACGC GAGCGAACGT 8580 GAAGCGACTGCTGCTGCAAA ACGTCTGCGA CCTGAGCAAC AACATGAATG GTCTTCGGTT 8640 TCCGTGTTTCGTAAAGTCTG GAAACGCGGA AGTCAGCGCC CTGCACCATT ATGTTCCGGA 8700 TCTGCATCGCAGGATGCTGC TGGCTACCCT GTGGAACACC TACATCTGTA TTAACGAAGC 8760 CTTTCTCAATGCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG 8820 GGCTGTGTGCACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT 8880 CTTGAGTCCAACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG 8940 ATTAGCAGAGCGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC 9000 GGCTACACTAGAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA 9060 AAAAGAGTTGGTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT 9120 GTTTGCAAGCAGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT 9180 TCTACGGGGTCTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA 9240 TTATCAAAAAGGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC 9300 TAAAGTATATATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT 9360 ATCTCAGCGATCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA 9420 ACTACGATACGGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA 9480 CGCTCACCGGCTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA 9540 AGTGGTCCTGCAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA 9600 GTAAGTAGTTCGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTGC AGGCATCGTG 9660 GTGTCACGCTCGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA 9720 GTTACATGATCCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT 9780 GTCAGAAGTAAGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT 9840 CTTACTGTCATGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA 9900 TTCTGAGAATAGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAC ACGGGATAAT 9960 ACCGCGCCACATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA 10020 AAACTCTCAAGGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC 10080 AACTGATCTTCAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG 10140 CAAAATGCCGCAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC 10200 CTTTTTCAATATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT 10260 GAATGTATTTAGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA 10320 CCTGACGTCTAAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG 10380 AGGCCCTTTCGTCTTCAA 10398 30 base pairs nucleic acid single unknown other nucleicacid 2 GGCCTCTAGA TGGAGGGGTG GAGTCGTGAC 30 30 base pairs nucleic acidsingle unknown other nucleic acid 3 GGCCGGATCC AACGCGCAGC CGCCATGCCG 3030 base pairs nucleic acid single unknown other nucleic acid 4GGCCGGATCC CAAACCTCCC GCTTCAAAAT 30 30 base pairs nucleic acid singleunknown other nucleic acid 5 GGCCGAGCTC AGGCTGGGTT TTGGGGAGCA 30

What is claimed is:
 1. A method for production of recombinantadeno-associated virus (AAV) comprising culturing a host cellcomprising: (a) a recombinant adenovirus comprising a cre gene undercontrol of sequences which permit expression of cre recombinase; (b) anucleic acid molecule comprising a spacer sequence flanked by lox sites,and AAV rep and cap genes, wherein the spacer sequence is upstream ofthe AAV genes; and (c) a minigene comprising a transgene flanked by AAVinverted terminal repeats (ITRs); in the presence of helper virusfunctions which permit packaging of the minigene into an AAV capsid,whereby a recombinant AAV capable of expressing said transgene isproduced.
 2. The method according to claim 1, comprising: (a)introducing into a host cell (i) the recombinant adenovirus; (ii) asecond vector comprising from 5′ to 3′, a selected promoter, a spacersequence flanked by loxP sites, and AAV rep and AAV cap genes; (iii) athird vector comprising a minigene consisting essentially of, from 5′ to3′, a 5′ AAV ITR, a promoter, a transgene and 3′ AAV ITR; (b) culturingthe host cell under conditions which permit expression of the crerecombinase; and (c) recovering recombinant AAV capable of expressingthe product of said transgene.
 3. The method according to claim 1wherein in the recombinant adenovirus the sequences which permitexpression comprise a cytomegalovirus promoter, and the adenovirusfurther comprises a nuclear localization signal operably linked to thecre gene.
 4. The method according to claim 1 wherein the spacer sequenceis selected from the group consisting of: (a) a 1300 bp fragmentcontaining translational start and stop sequences; (b) a 1600 bpfragment containing the green fluorescent protein (GFP) cDNA, an intronand a polyadenylation signal; and (c) a 1000 bp fragment containing theneomycin coding sequence and a polyadenylation signal.
 5. The methodaccording to claim 2 wherein at least one of said vectors is arecombinant adenovirus and the host cell is a 293 cell.
 6. The methodaccording to claim 2 wherein the second vector is a recombinantadenovirus and comprises an AAV P5 promoter.